1. Field of the Invention
The present invention relates generally to the fields of molecular biology. More particularly, it concerns methods for affecting and/or altering the differentiation state of a cell. In certain embodiments, the present invention provides a method to transdifferentiate a cell such as fibroblast or a fibroblast like cell, into a vascular endothelial cell, hematopoietic cell, bone marrow-derived fibroblastic cells, T cells, cells of the granulocyte and monocyte lineage and B cells.
2. Description of the Related Art
Approximately 30 percent of patients who require hematopoietic stem cell transplantation will identify a suitably matched unrelated donor through the National Marrow Donor Program. However, the patient's disease may progress and worsen during the approximately 2 months required to identify an acceptably matched unrelated donor and obtain the graft. This time period would reduce or eliminate the chance of success. In other words, during this waiting period some patients will succumb to the disease and, in many others, the disease will progress to a state at which the patients will not be eligible to undergo hematopoietic stem cell transplantation. For those individuals who require an urgent hematopoietic transplant (most patients) and others for whom an unrelated donor search is unsuccessful, the rapid availability of a partially matched umbilical cord blood (UCB) unit from a volunteer donor cord blood bank has been suggested to offer an alternative treatment option. It has been estimated that approximately 7200 allogeneic hematopoietic transplants were performed in the United States during 2002, and nearly double that number were performed worldwide. More than 200,000 UCB units are available to the public from cord blood banks (1), and more than 3,500 unrelated donor hematopoietic transplants have been performed with UCB; UCB grafts represented a significant and growing fraction of the sources of all hematopoietic grafts employed for pediatric patients in the year 2002 (approximately 15%) (Source: National Marrow Donor Program, Minneapolis (Minn.)).
There are, however, controversies surrounding the role of UCB grafts in hematopoietic transplantation and several disadvantages have been identified. Those include 1) the relative paucity of progenitor cells available (the minimal acceptable cell dose for single unit UCB for adults varies, but is typically at least 1.5×107 to 2.0×107 per kg, and some studies have suggested that a higher minimal doses is needed), 2) prolonged time of neutrophil and platelet engraftment, 3) a higher rate of engraftment failure, 4) concerns about reduced immunity to infections, 5) reduced anti tumor activity, 6) inability to obtain additional cells from the donor, 7) the difficulty in searching the large number of existing cord blood banks for matched grafts, 8) concerns regarding variations in the required handling and preparation of the units, 9) uncertain expectations for cell recovery after thawing, and 10) concerns regarding the overall quality of UCB among the cord blood banks (2). Hematopoietic stem cell transplantation using umbilical cord blood progenitors in patients is currently being experimented in the clinic. Taken together, there is a clear and urgent need to identify another easily accessible cellular source suitable for hematopoietic transplantation.
The non-hematopoietic component of a normal bone marrow consists of several cell types. Friedenstein (3) was the first investigator who described clonal, fibroblast-like plastic adherent cells from bone marrow capable of differentiating into osteoblasts, adipocytes, and chondrocytes (4-7). These cells, later termed mesenchymal stem cells (MSC), are also stromal cells; the structural components of the bone marrow that support ex vivo culture of hematopoesis by providing extracellular matrix components, cytokines, and growth factors (3,8-12). Numerous investigators have now demonstrated that multipotent MSCs can be recovered from a variety of adult tissues and differentiate into a variety of tissue lineages including myoblasts, hepatocytes, and possibly even neural tissue (13-16). One group has reported that hematopoietic stem cells (HSCs) can be differentiated into multiple blood lineages through the application of a demethylation agent (5aza 2′deoxycytidine) and a deacetylation inhibitor (trichostatin A) (Milhem et al., 2004; US 2005/0276793).
The question of how a single cell can differentiate into the many different cell types has long led to the postulation that additional information that regulates genomic functions must exist beyond the level of the genetic code. This concept led to the introduction of the term “epigenetics” back in the 1940s—a term that has now evolved to mean heritable changes in gene expression that do not involve changes in DNA sequence (32). Epigenetic regulation is not only critical for generating diversity of cell types during mammalian development, but it is also important for maintaining the stability and integrity of the expression profiles of different cell types. Interestingly, whereas these epigenetic changes are heritable and normally stably maintained, they are also potentially reversible, as evidenced by the success of cloning entire organisms by nuclear transfer methods using nuclei of differentiated cells (33).
Studies of the molecular basis of epigenetics have largely focused on mechanisms such as DNA methylation and chromatin modifications (34). In fact, emerging evidence indicates that both mechanisms act in concert to provide stable and heritable silencing in higher eukaryotic genomes. DNA methylation is a biochemical modification that, in human cells, primarily affects cytosines when they are part of the symmetrical dinucleotide CpG. Cytosine methylation has long been a challenging scientific puzzle. In mammals, DNA methylation is essential for normal development, but its evolutionary raison d'être remains controversial. A commonly held hypothesis is that DNA methylation originally evolved to silence repetitive elements, and that this silencing property has also been put to use in other situations where transcriptional silencing is required, such as imprinting (a process whereby one of the two alleles of a gene are permanently inactivated, depending on which parent that allele was inherited from) and X-chromosome inactivation. Most CpG sites have been lost from mammalian genomes during evolution, but about 1% of human DNA consists of short areas where CpG sites have escaped depletion. Most of the remaining CpG sites are normally methylated in adult cells. About half of all genes have a CpG island in their promoter region, and this gene configuration is what has recently attracted the most attention. Most promoter CpG islands are normally unmethylated, regardless of the expression state of the associated gene. However, in silenced areas, such as the inactive X-chromosome in females and the silenced allele of imprinted genes, promoter-associated CpG islands are generally methylated, and this methylation is essential for maintaining the silenced state.
Mechanisms regulating the establishment of methylation remain poorly understood, but the consequences of CpG island methylation are becoming increasingly clear. Methylation triggers the binding of methylated DNA-specific binding proteins to CpG sites, attracting histone-modifying enzymes that, in turn, focally establish a silenced chromatin state. Consistent with a resurgence of interest in the idea that cancer is a disease of faulty development, there has been a revival of interest in the epigenetic processes involved in neoplastic development and progression. The potential reversibility of epigenetic changes through pharmacological manipulation makes this area important in cancer management, and specific DNA methylation inhibitors are currently being used as anti-cancer agents in the USA: 5-Azacytidine has now been approved for use and decitabine proved to be clinically effective in myelodysplastic syndrome and myeloid leukemias (35-37).
Mesenchymal and mesenchymal-like cells (found by the inventors to be applicable to the present invention) have been recovered from a rapidly expanding list of tissues. Exemplary cells can also be recovered from human fat aspirates (38), cryo-preserved human umbilical cord blood (39), placental tissue (40) and (41), and even human exfoliated deciduous teeth (42). Furthermore, a multipotent precursor cell from mammalian dermis that can differentiate into both neural and mesodermal progeny has previously isolated, expanded, and characterized (43, 44), and later this group reported the isolation, expansion, and characterization of a similar precursor cell from neonatal human foreskin tissue. Like their rodent counterparts, the human skin cells grew in suspension as spheres in the presence of the growth factors, fibroblast growth factor 2 and epidermal growth factor and expressed several adhesion molecules and characteristic embryonic transcription factors. These human skin cells could be maintained in culture for long periods of time induce to differentiate into neurons, glia, and smooth muscle cells (45). Most recently, Bartsch et al. isolated MSCs from human postnatal dermal tissues. The isolated cells were expanded and maintained for over 100 population doublings with retention of their chromosomal complement and potential for multilineage differentiation.
Progeny of cell lines established from a single dermal mesenchymal cell could be differentiated into adipogenic, osteogenic, and myogenic lineages, consistent with the conclusion that they established a clonal, multipotential, somatic mesenchymal cell line (46). Because multipotent mesenchymal cells are easily expanded in culture and differentiate into several tissue lineages, there has been much interest in their clinical potential for tissue repair and gene therapy (47). Numerous laboratories have now demonstrated that mesenchymal and mesenchymal-like cells (such as bone marrow derived fibroblastic cells) recovered from a variety of adult tissues differentiate into various tissue lineages in vitro. In particular, Verfaille and colleagues, report that a specific type of murine mesenchymal cells isolated from bone marrow, muscle, or brain, termed multipotential adult progenitor cells (MAPCs), differentiates into a variety of tissue lineages including myoblasts, hepatocytes, and even neural tissue (13-16).